The present invention concerns bioelastic polymers and methods of use thereof, particularly for immunoassays, for the production of regenerable biosensors, for the localized delivery of compounds in vivo, and for the patterning of molecules on substrates.
Numerous bioelastic polymers, also known as bioelastomers or elastin-like proteins (xe2x80x9cELPsxe2x80x9d) are known. Examples are described in D. Urry et al., A Simple Method for the Purification of a Bioelastic Polymer, PCT Application WO 96/32406. Such compounds are proteins or peptides, typically polypeptides, that exhibit an inverse temperature transition: that is, the compounds condense at a higher temperature range in an aqueous system on raising the temperature of the compounds through their transition temperature (TI). Bioelastic polymers are soluble in water at a sufficiently low temperature, but hydrophobically fold and associate to form a separate phase as the temperature is raised through a particular temperature range.
Immunoassays are commonly used to detect analytes such as enzymes, hormones, drugs, and other molecules of interest in complex biological mixtures. By definition, an immunoassay relies on the specific binding of an antigen by an antibody, but assays using other biological high affinity binding partners (e.g., ligand-cell surface receptor, inhibitor-enzyme, etc.) can also be employed in an analogous manner. A number of different immunoassay formats have been developed to detect and/or quantitate the levels of analytes; for a review of immunoassays, see PCT Patent Application WO 86/06492.
Ligand-binding proteins such as receptors and antibodies, currently used in biosensors, can detect specific analytes (ligands) with high sensitivity in the presence of potential interference""s in complex mixtures. The high affinity of protein-analyte interactions is the basis of their exquisite sensitivity. However, high affinity is generally accompanied by an extremely slow dissociation rate (off rate) of the protein-ligand complex. Therefore, in practice, most biosensors are xe2x80x9cone shotxe2x80x9d devices; dosimeters rather than continuous sensors or alternatively, sensors with very slow response times. In order to use biosensors for the semi-continuous, in situ monitoring of analytes, or for subsequent rounds of sensing in batch mode, the sensor must be regenerated for reuse in an expedient time frame.
There are two possible approaches to sensor regeneration. When the receptor is covalently coupled to the sensor surface, free receptor can be regenerated by displacing the bound analyte. Unfortunately, methods to gently and reversibly regenerate analyte-free receptor do not currently exist: most current methods disrupt noncovalent interactions between analyte and receptor by partially denaturing the receptor using drastic changes in the protein-ligand environment such as low pH ( less than 3), or high chaotrope concentration, conditions which often irreversibly denature the protein after a few rounds of regeneration.
If the receptor is not covalently attached to the substrate, a second method for surface regeneration is feasible where the surface itself can be regenerated by removing the analyte-bound receptor from the surface. The potential advantage is that the analyte xe2x80x9cseesxe2x80x9d fresh receptor in every round of sensing, which can decrease drift in the sensor response and maintain high affinity and homogenous binding kinetics. This approach to sensor regeneration is difficult to broadly implement because noncovalent methods to immobilize proteins on surfaces typically involves their physical adsorption, which is typically irreversible, and subsequent stripping of adsorbed protein with detergents or chaotropes is frequently incomplete. In order to noncovalently and reversibly bind a receptor to the surface, methods must be found to reversibly control the physico-chemical properties of the receptor such that the adsorption-desorption process can be triggered reversibly.
The targeted delivery of drugs to solid tumors is a complex problem because of the impediments to drug delivery that are posed by tumor heterogeneity. Cancer cells typically occupy less than half of the total tumor volume. Approximately 1-10% is contributed by tumor vasculature, and the rest is occupied by a collagen-rich interstitium. The major impediments to drug delivery arise from heterogeneous distribution of blood vessels, combined with aberrant branching and tortuosity, which results in uneven and slowed blood flow. The leakiness of tumor vessels combined with the absence of a functional lymphatic system results in an elevated interstitial pressure, which retards the convective transport of high MW ( greater than 2000 Da) drugs. R. Jain, Sci. Am. 271: 58-65 (1994). The heterogeneity of antigen and receptor expression in tumors is an additional problem in affinity-targeted delivery of drugs to solid tumors.
Front-line therapies for different tumors include surgery, chemotherapy, and radiation. The infiltrative nature of many solid tumors often prevents complete surgical resection because of the high risk of compromising function, thereby necessitating postoperative chemotherapy and/or radiotherapy. However, chemotherapy, particularly when delivered systemically is of limited effectiveness due to inadequate drug delivery, systemic toxicity, and a markedly variable biological sensitivity. External beam irradiation, while useful for many types of tumors, is also limited by dose limiting toxicity to healthy tissue.
Two other treatment modalities that have been suggested for the treatment of solid tumors, are hyperthermia [S. Field and J. Hand, An Introduction to the Practical Aspects of Clinical Hyperthermia (Taylor and Francis, London 1990)] and targeted radiotherapy [C. Hoefnagel., Int. J. Biol. Markers 8: 172 (1993); M. Gaze, Phys. Med. Biol. 41: 1895 (1993)]. The use of local hyperthermia as a therapeutic modality for sold tumors is motivated by the increased thermal sensitivity of tumor vasculature compared to normal vasculature. Hyperthermia, at temperatures between 40 and 42xc2x0 C., is known to increase tumor blood flow and vascular permeability. Because hyperthermia sensitizes cells to radiation, it has been combined with radiation therapy to increase tumor cytotoxicity [M. Hauck et al., in Handbook of Targeted Delivery of Imaging Agents, pp. 335-361 (V. Torchilin Ed. 1995)].
The limitations of current therapeutic approaches for the management of solid tumors provide a compelling need for the development of improved modalities for the targeted delivery of therapeutics.
A first aspect of the present invention is a method of binding a compound of interest in an aqueous solution. The method comprises the steps of:
(a) providing a conjugate comprising a bioelastic compound and a binding compound, wherein the binding compound specifically binds the compound of interest, and wherein the bioelastic compound has a transition temperature below which the bioelastic compound is soluble in the solution, and above which the bioelastic compound is insoluble in the solution;
(b) contacting the conjugate to the compound of interest in an aqueous solution so that the compound binds thereto, with the contacting step carried out at a temperature below the transition temperature of the bioelastic compound; and then
(c) raising the temperature of the conjugate to a temperature above the transition temperature of the bioelastic compound so that the conjugate separates from the aqueous solution with the compound of interest bound thereto. Separation may be passive (e.g., precipitation in solution) or active (e.g., by centrifugation or filtration). Thus, the step of xe2x80x9craising the temperaturexe2x80x9d may be thought of as being followed by a step of separating (i.e., actively separating) the conjugate with the compound of interest bound thereto from the solution.
A second aspect of the present invention is a method useful for immunologically detecting an analyte in an aqueous solution. The method comprises the steps of:
(a) providing a conjugate comprising a bioelastic compound and a binding compound, wherein the binding compound specifically binds the analyte, and wherein the bioelastic compound has a transition temperature below which the bioelastic compound is soluble in the solution, and above which the bioelastic compound is insoluble in the solution;
(b) contacting the conjugate to the analyte in an aqueous solution so that the compound binds thereto, with the contacting step carried out at a temperature below the transition temperature of the bioelastic compound;
(c) raising the temperature of the conjugate to a temperature above the transition temperature of the bioelastic compound so that the conjugate separates from the aqueous solution with the analyte bound thereto; and then
(d) detecting the analyte.
A third aspect of the present invention is an article useful as a regenerable biosensor for binding a compound of interest from an aqueous solution, or for any such other purposes to which the article may be suitable. The article comprises:
(a) a solid support having a hydrophobic surface formed thereon; and
(b) a conjugate reversibly bonded to the hydrophobic surface, the conjugate comprising (i) a bioelastic compound and a (ii) binding compound, wherein the binding compound specifically binds the compound of interest, and wherein the bioelastic compound has a transition temperature below which the bioelastic compound is soluble in the solution, and above which the bioelastic compound is insoluble in the solution; so that the biosensor may be used to bind the compound of interest at a temperature above the transition temperature, and so that the conjugate can be removed from the solid support for recycling of the article by lowering the temperature of the solid support (and/or the solution; so long as the temperature of the conjugate is lowered) below the transition temperature.
A fourth aspect of the invention is a method of recycling a used biosensor to which has been bound a compound of interest. The method comprises:
(a) providing a biosensor comprising a solid support having a hydrophobic surface formed thereon and a first conjugate reversibly bonded to the hydrophobic surface, the conjugate comprising (i) a bioelastic compound (which is bonded to the hydrophobic surface by hydrophobic interactions when at a temperature above its transition temperature) and (ii) a binding compound, wherein the binding compound has the compound of interest specifically bound thereto, and wherein the bioelastic compound has a transition temperature below which the bioelastic compound is soluble in the solution, and above which the bioelastic compound is insoluble in the solution; and then
(b) separating the conjugate with the compound of interest bound thereto from the solid support by lowering the temperature of the biosensor to below the transition temperature; and then
(c) binding a second conjugate to the hydrophobic surface, the second conjugate comprising a second bioelastic compound and a second binding compound, so that the biosensor may be reused. The second conjugate may be the same as or different from the first conjugate.
A fifth aspect of the present invention is a method for the targeted delivering of a compound in vivo to a selected region within a subject. The method comprises:
(a) administering a conjugate to the subject, the conjugate comprising the compound to be delivered and a polymer that undergoes an inverse temperature transition, wherein the polymer has a transition temperature (TI) greater than the temperature at which the compound is delivered; and then
(b) heating the selected region to a temperature greater than the transition temperature of the polymer, so that the compound is preferentially delivered to the selected region.
A sixth aspect of the present invention is the use of a conjugate as described above for the preparation of a medicament for the targeted delivery of a compound as described above.